The ability of cartilage to act as a shock absorber depends on the quality of the collagen fibrillar network that frames cartilage. This network is made up of three different types of collagen (types II, IX and XI) assembled and precisely cross-linked together into heteropolymers. Despite many advances in the field of cartilage tissue engineering, a continuous challenge has been to increase the collagen content of bioengineered cartilage to levels observed in native articular cartilage. Since the collagen heteropolymer is a crucial template of the mature fibrillar architecture and for the ongoing stability of the mature tissue, we hypothesize that a cross-linked template of type II/IX/XI collagen heterofibrils must sequentially assemble in the matrix of newly forming cartilage to allow type II collagen fibril growth, an increased collagen content and so achieve the biomechanical properties of functional articular cartilage. Whether correct cross-linked assemblies of the minor collagen template (types IX and XI) with type II collagen form in neo-cartilage, is not well characterized. Our goal is to determine if a collagen network typical of hyaline cartilage is assembled in chondrocyte based and mesenchymal stem cell based bio-engineered tissues. Since the minor collagens regulate the organization of the network, (e.g., collagen fibril diameter modulated by type XI/V collagen), fingerprinting the pattern of collagen inter-type cross-linking can provide a screen for normal matrix assembly and a valuable tool in understanding the sequence of events in the fibrillogenesis of the type II/IX/XI collagen fibril in adult mesenchymal stem cells undergoing chondrogenesis. The aim is to use Western blot, ELISA, LC-mass spectrometry, and other advanced methods in protein chemistry to establish a molecular fingerprint of the collagen fibril assembly and a set of parameters to use as a biomarker to better evaluate the cross-linked collagen network in normal and engineered cartilage. The ultimate goal is to validate proteomic methods to use as an outcome measure of tissue engineered cartilage that has the collagen content, quality and structural properties of articular cartilage needed to transplant into cartilage defects. The goals also include laying the foundation for evaluating the changing quality of cartilage produced as a healing response. PUBLIC HEALTH RELEVANCE: The ability of cartilage to act as a shock absorber depends on the quality of the collagen heterofibrillar network that frames cartilage. In arthritis, this collagen network is broken down leading to a loss of normal joint function and severe pain. The goal of this study is to develop and authenticate a biomarker to monitor if this collagen heterofibrillar network assembled in bioengineered cartilage is typical of normal cartilage.